A. Bruce Lyons

Analysing cell division in vivo and in vitro using flow cytometric measurement of CFSE dye dilution.

Since its introduction in 1994 (J. Immunol. Methods 171 (1994) 131), the flow cytometric analysis of lymphocyte proliferation by serial halving of the fluorescence intensity of the vital dye CFSE (carboxyfluorescein diacetate, succinimidyl ester or CFDA-SE) has become widely used in immunological laboratories around the world. This technique allows the visualisation of eight to 10 discrete cycles of cell division by flow cytometry, both in vitro and in vivo. Appropriately conjugated antibodies can be used to probe surface marker changes as cells divide, or changes in expression of internal molecules such as cytokines when appropriate fixation and permeabilisation protocols are used. An added advantage of the technique is the ability to recover viable cells which have undergone defined numbers of cell divisions by flow cytometric sorting, allowing functional studies to be performed. Other commonly used assays of cell proliferation give only limited information, as they usually measure division at a population level. The CFSE technique can be used to determine kinetics of immune responses, track proliferation in minor subsets of cells and follow the acquisition of differentiation markers or internal proteins linked to cell division.

Cell division number regulates IgG1 and IgE switching of B cells following stimulation by CD40 ligand and IL-4

CD40 ligand (CD40L) and IL‐4 are sufficient to induce resting murine B cells to divide and switch isotypes from IgM and IgD to IgG1 and IgE. Tracking of cell division following (5‐and 6) carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling revealed that B cells expressed IgG1 after three cell divisions, and IgE after five. The probability of isotype switching at each division was independent of both time after stimulation and of the dose of CD40L. IL‐4 concentration regulated the number of divisions that preceded isotype switching. Loss of surface IgM and IgD was also related to cell division and appeared to be differentially regulated. B cell proliferation was typically asynchronous with the proportion of cells in consecutive divisions being markedly affected by the concentration of CD40L and IL‐4. Simultaneous (5‐bromo)‐2 ′ ‐deoxyuridine labeling and CFSE staining revealed that B cells in each division cycle were dividing at the same rate. Therefore, division cycle asynchrony resulted from dose‐dependent variation in the time taken to enter the first division cycle. These results suggest that T‐dependent B cell expansion is linked to predictable functional changes that may, in part, explain why IgE is produced in response to prolonged antigenic stimulation.

Dasatinib suppresses in vitro natural killer cell cytotoxicity

To the editor: The recent publication by Schade et al[1][1] demonstrated that the Src/Abl kinase inhibitor dasatinib (Sprycel), used for the treatment of chronic myeloid leukemia,[2][2] inhibits the function of T cells in vitro and in vivo. Dasatinib inhibition of the Src-family kinase LCK, which

A second transmissible cancer in Tasmanian devils

Significance Transmissible cancers are somatic cell lineages that are spread between individuals via the transfer of living cancer cells. Only three transmissible cancers have been reported in nature, suggesting that such diseases emerge rarely. One of the known transmissible cancers affects Tasmanian devils, and is threatening this species with extinction. Here we report the discovery of a second transmissible cancer in Tasmanian devils. This cancer causes facial tumors that are grossly indistinguishable from those caused by the first-described transmissible cancer in this species; however, tumors derived from this second clone are genetically distinct. These findings indicate that Tasmanian devils have spawned at least two different transmissible cancers, and suggest that transmissible cancers may arise more frequently in nature than previously considered. Clonally transmissible cancers are somatic cell lineages that are spread between individuals via the transfer of living cancer cells. There are only three known naturally occurring transmissible cancers, and these affect dogs, soft-shell clams, and Tasmanian devils, respectively. The Tasmanian devil transmissible facial cancer was first observed in 1996, and is threatening its host species with extinction. Until now, this disease has been consistently associated with a single aneuploid cancer cell lineage that we refer to as DFT1. Here we describe a second transmissible cancer, DFT2, in five devils located in southern Tasmania in 2014 and 2015. DFT2 causes facial tumors that are grossly indistinguishable but histologically distinct from those caused by DFT1. DFT2 bears no detectable cytogenetic similarity to DFT1 and carries a Y chromosome, which contrasts with the female origin of DFT1. DFT2 shows different alleles to both its hosts and DFT1 at microsatellite, structural variant, and major histocompatibility complex (MHC) loci, confirming that it is a second cancer that can be transmitted between devils as an allogeneic, MHC-discordant graft. These findings indicate that Tasmanian devils have spawned at least two distinct transmissible cancer lineages and suggest that transmissible cancers may arise more frequently in nature than previously considered. The discovery of DFT2 presents important challenges for the conservation of Tasmanian devils and raises the possibility that this species is particularly prone to the emergence of transmissible cancers. More generally, our findings highlight the potential for cancer cells to depart from their hosts and become dangerous transmissible pathogens.

Flow Cytometric Analysis of Cell Division by Dye Dilution

The technique described in this unit uses the intracellular fluorescent label carboxyfluorescein diacetate succinimidyl ester (CFSE) to tag proliferating cells. Covalently bound CFSE is divided equally between daughter cells, allowing discrimination of successive rounds of cell division. The technique is applicable to in vitro cell division as well as to in vivo division of adoptively transferred cells and can resolve up to eight successive generations. CFSE is long lived, permitting analysis for several months after transfer, and has the same spectral characteristics as fluorescein, so monoclonal antibodies conjugated to phycoerythrin or other compatible fluorochromes may be used to immunophenotype the dividing cells.

Dasatinib inhibits recombinant viral antigen-specific murine CD4+ and CD8+ T-cell responses and NK-cell cytolytic activity in vitro and in vivo

OBJECTIVE Dasatinib (BMS-354825) is a small molecule Src/Abl tyrosine kinase inhibitor approved for the treatment of chronic myeloid leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia. Members of the Src family of kinases are involved in the induction of innate and adaptive immunity. The purpose of this study was to evaluate the inhibitory action of dasatinib on antigen-specific CD8(+) and CD4(+) T-cell function, as well as natural killer (NK) cell cytotoxicity. MATERIALS AND METHODS To assess dasatinib-mediated inhibition of antigen-specific T-cell proliferation, transgenic CD4(+) and CD8(+) T cells specific for ovalbumin were utilized. Endogenous CD4(+) and CD8(+) T-cell responses were determined following immunization of dasatinib-treated or control mice with a nonreplicating recombinant virus. Clearance of the RMA-S cells, a major histocompatibility complex (MHC) class I-deficient thymoma sensitive to NK-cell lysis, was analyzed in mice undergoing dasatinib treatment. RESULTS Dasatinib inhibited antigen-specific proliferation of murine CD4(+) and CD8(+) transgenic T cells in vitro and in vivo. Endogenous antigen-specific helper T-cell recall responses and induction of T-cell-mediated cytotoxicity following immunization with a nonreplicating recombinant virus were also inhibited. So to was the ability of NK cells to eliminate MHC class I-deficient cells in vivo. CONCLUSIONS These findings suggest that dasatinib has the potential to modulate the host immune response at clinical doses and highlights scope for off target applications, e.g., therapeutic immunosuppression in the context of autoimmune pathogenesis and allogeneic tissue transplantation.

Imatinib inhibits the functional capacity of cultured human monocytes

Imatinib is a tyrosine kinase inhibitor that has been reported to specifically inhibit the growth of bcr‐abl expressing chronic myeloid leukaemia progenitors. This drug functions by blocking the ATP‐binding site of the kinase domain of bcr‐abl, and has also been found to inhibit the c‐abl, platelet‐derived growth factor receptor, ARG and stem cell factor receptor tyrosine kinases. Reports have recently emerged demonstrating that imatinib also inhibits the growth of non‐malignant haemopoietic cells. Here, we demonstrate that concentrations of imatinib within the therapeutic dose range inhibit the function of cultured monocytes (CM) from normal donors. A decrease in the response of CM to LPS was observed morphologically and functionally, with CM grown in the presence of imatinib showing decreased pseudopodia formation and inhibition of IL‐6 and TNF‐α production following LPS stimulation. Imatinib also reduced the ability of M‐CSF and GM‐CSF stimulated CM to phagocytose zymosan particles, with uptake of non‐opsonized zymosan by M‐CSF stimulated CM (M‐CM) being most affected. M‐CM that had been cultured in the presence of imatinib were also impaired in their ability to stimulate responder cells in a mixed lymphocyte reaction. These results demonstrate that human monocytes cultured in the presence of imatinib are functionally impaired, and suggest that imatinib displays inhibitory activity against other kinase(s) that play a role in monocyte/macrophage development.

Flow Cytometric Analysis of Cell Division by Dilution of CFSE and Related Dyes.

The technique described in this unit uses the intracellular fluorescent label carboxyfluorescein diacetate succinimidyl ester (CFSE) to track proliferating cells. Covalently bound CFSE is divided equally between daughter cells, allowing discrimination of successive rounds of cell division. The technique is applicable to in vitro cell division, as well as to in vivo division of adoptively transferred cells and can resolve eight or more successive generations. CFSE is long lived, permitting analysis for several months after cell transfer, and has the same spectral characteristics as fluorescein, so monoclonal antibodies conjugated to phycoerythrin or other compatible fluorochromes may be used to immunophenotype the dividing cells. In addition, information is given on a second‐generation dye, Cell Trace Violet (CTV), excited by 405‐nm blue laser light. CTV is chemically related to CFSE, but allows the 488‐nm line of the Argon laser to be used for other probes. Curr. Protoc. Cytom. 64:9.11.1‐9.11.12.

Acquisition of immune function during the development of the Langerhans cell network in neonatal mice

The immunological function of the Langerhans cell (LC) network in neonatal skin was examined by defining the development of cutaneous immunity relative to the structure, phenotype and function of the epidermal LC network in neonatal, juvenile and adult mice. Analysis of epidermal sheets showed the presence of major histocompatibility complex (MHC) II+, multilectin receptor DEC‐205– cells within the epidermis of 3‐day‐old mice; both cell density and DEC‐205 expression increased until day 14. When visualized with antibodies directed at MHC II, the network was poorly formed in 3‐ and 7‐day‐old mice, as there was a lower cell density and poor MHC II expression on dendritic processes, compared to mice at day14. Application of a fluorescent antigen to 3‐day‐old mice revealed that the LC were inefficient in transporting antigen to the draining lymph node. There was an improvement at day 7 and by day 14 comparable numbers of antigen carrying cells were detected in the lymph nodes of 6‐week‐old mice. The reduced antigen carriage in 3‐ and 7‐day‐old mice correlated with a poor contact sensitivity response. This was not simply due to failure to present antigen, but development of immunosuppression, as transfer of T cells from adult mice that were previously treated with antigen when they were 3 days old, to adult recipients resulted in antigen specific immunosuppression. Analysis of CD80 and CD86 expression showed that LC from day 3 skin expressed CD80, but not CD86 and application of antigen through this skin was inefficient in upregulating CD86. These findings indicate that when the neonatal LC network is poorly developed it is functionally immature and antigen applied through this ‘functionally immature network’ results in antigen specific immunosuppression.

Natural Killer Cell Mediated Cytotoxic Responses in the Tasmanian Devil

The Tasmanian devil (Sarcophilus harrisii), the worlds largest marsupial carnivore, is under threat of extinction following the emergence of an infectious cancer. Devil facial tumour disease (DFTD) is spread between Tasmanian devils during biting. The disease is consistently fatal and devils succumb without developing a protective immune response. The aim of this study was to determine if Tasmanian devils were capable of forming cytotoxic antitumour responses and develop antibodies against DFTD cells and foreign tumour cells. The two Tasmanian devils immunised with irradiated DFTD cells did not form cytotoxic or humoral responses against DFTD cells, even after multiple immunisations. However, following immunisation with xenogenic K562 cells, devils did produce cytotoxic responses and antibodies against this foreign tumour cell line. The cytotoxicity appeared to occur through the activity of natural killer (NK) cells in an antibody dependent manner. Classical NK cell responses, such as innate killing of DFTD and foreign cancer cells, were not observed. Cells with an NK-like phenotype comprised approximately 4 percent of peripheral blood mononuclear cells. The results of this study suggest that Tasmanian devils have NK cells with functional cytotoxic pathways. Although devil NK cells do not directly recognise DFTD cancer cells, the development of antibody dependent cell-mediated cytotoxicity presents a potential pathway to induce cytotoxic responses against the disease. These findings have positive implications for future DFTD vaccine research.